RNA-based, amplification-free, organism identification using nano-enabled electronic detection

ABSTRACT

A technique that uses nanotechnology to electrically detect and identify RNA sequences without the need for using enzymatic amplification methods or fluorescent labels. The technique may be scaled into large multiplexed arrays for high-throughput and rapid screening. The technique is further able to differentiate closely related variants of a given bacterial or viral species or strain. This technique addresses the need for a quick, efficient, and inexpensive bacterial and viral detection and identification system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/675,958 filed on Apr. 1, 2015, incorporated herein by reference inits entirety, which is a 35 U.S.C. § 111(a) continuation of PCTinternational application number PCT/US2013/063496 filed on Oct. 4,2013, incorporated herein by reference in its entirety, which claimspriority to, and the benefit of, U.S. provisional patent applicationSer. No. 61/709,765 filed on Oct. 4, 2012, incorporated herein byreference in its entirety. Priority is claimed to each of the foregoingapplications.

The above-referenced PCT international application was published as PCTInternational Publication No. WO 2014/055888 on Apr. 10, 2014, whichpublication is incorporated herein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

This application includes a sequence listing in a text file entitled“UC-2013-028-3-US-div-testsequence13-ST25.txt” created on Sep. 12, 2017and having a 1 kb file size. The sequence listing is submitted throughEFS-Web and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention pertains generally to a system and methods for detectingand identifying RNA sequences in biological organisms, and moreparticularly to a technique that uses nanotechnology to electricallydetect and identify RNA, including but not limited to bacterial andviral coding and non-coding RNA sequences, without the necessity ofusing enzymatic amplification methods or fluorescent markers.

2. Description of Related Art

Rapid, efficient, and low cost detection and identification of anorganism based on its nucleic acid is a challenge facing those who carefor plant and animal health. Current technologies, such as quantitativepolymerase chain reaction (q-PCR), rely on multiple assays andamplification methods to identify organisms based on collected nucleicacid. Traditional optical detection methods also require fluorescentmarkers. These multiple independent steps and tests increase theprocessing time and cost of detection and identification. There is aclear and evident need to develop new technologies that can quickly,efficiently, and inexpensively identify organisms using their nucleicacid, especially microorganisms associated with plants and humans, basedon genetic markers.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention provides a system and method thatuses nanotechnology to electrically detect and identify an organismusing RNA sequences without the need for enzymatic amplification methodsor fluorescent markers. Although the present invention is describedherein with reference to microorganisms, the present invention isapplicable to detecting and identifying any biological organism based onthe organism's genetic information.

The present invention addresses the need for a quick, efficient, andinexpensive microbial detection and identification system that mayinclude, but is not limited to the detection and identification ofbacteria and viruses. In cases where microbe densities are particularlylow, the technique provides additional sensitivity that allows for thetarget molecules to be detected in small quantities. Furthermore, thetechnique may be scaled into large multiplexed arrays forhigh-throughput and rapid screening. The subject invention is furtherable to differentiate between closely related variants of a givenbacterial or viral species or strain.

An aspect of the invention is a method of precise and highly sensitivedetection and identification of bacteria and viruses in agricultural,medical, epidemiological, biosecurity, public health, and otherapplications.

Another aspect of the invention is to provide a platform thatelectrically detects genetic information (RNA) at the molecular levelwithout the use of fluorescent markers.

Another aspect of the invention is to provide a platform that removesthe need for enzymatic amplification (i.e., PCR).

Another aspect of the invention is to provide a platform for detectionand identification of specific species and strains.

Another aspect of the invention is to provide a platform that isamenable to multiplexing and facile integration with electronics forfield-deployable devices and high-throughput applications.

An embodiment of the present invention includes an electrical sensorplatform to detect bacteria and viruses using nanotechnology toelectrically detect RNA-based interactions for identification ofmicrobes that cause plant and human diseases.

In another embodiment, the present invention includes an electricalsensor platform to detect food-borne bacteria and plant viruses.

In another embodiment, the system includes an integrated sensor platformthat uses nanotechnology to electrically detect and identify coding andnon-coding RNA sequences without the necessity of enzymaticamplification methods (PCR). In one embodiment, the method uses thenatural amplification of RNA, and therefore, does not require the use ofenzymatic amplification.

Further aspects of the invention will be brought out in the followingportions of the specification, wherein the detailed description is forthe purpose of fully disclosing preferred embodiments of the inventionwithout placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The invention will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIG. 1 is a schematic diagram of an electrical sensor platform to detectbacteria and viruses according to one embodiment of the invention.

FIG. 2 is a schematic diagram of a single DNA:RNA hybrid bound betweentwo electrodes.

FIG. 3 shows the example probe sequence (N=13) SEQ. ID. NO. 1 used inexperiments.

FIG. 4 is a graph showing current vs. displacement traces for severalsingle molecule junctions including the sample sequence from FIG. 3.

FIG. 5 is a histogram resulting from a statistical analysis of curvessimilar to that shown in FIG. 4 for the N=9 length hybrid system.

FIG. 6 is a histogram resulting from a statistical analysis of curvessimilar to that shown in FIG. 4 for the N=13 length hybrid system.

FIG. 7 is a graph of conductance vs. length for the sequences studiedthus far.

FIG. 8A through FIG. 8D is a set of SEM images of nanoporous gold afterdifferent thermal treatments and the various morphologies caused byligament coarsening.

FIG. 9 is a graph showing the results of the surface area available forreaction when estimated electrochemically using dilute sulfuric acid asthe electrolyte.

FIG. 10 is a graph showing the amount of DNA immobilized as estimatedusing ruthenium hexamine chloride and potassium ferrocyanide as redoxmarkers.

DETAILED DESCRIPTION OF THE INVENTION

By way of example, and not of limitation, the present invention pertainsto RNA-based, amplification-free, organism identification usingnano-enabled electronic detection. The present invention furtherpertains to nano-molecular electronic detection of single molecule,RNA-based interactions for identification and characterization oforganisms including microbes causing plant, animal, and human diseases.Accordingly, the present invention could be used to identify infectiousdiseases or to identify specific genes being transcribed in mammals andplants.

In another embodiment of the present invention, the presence of singlenucleotide polymorphisms can be detected in a gene which can be used todetect genetic disorders and diseases.

In one embodiment, the method involves a two-tiered molecular baseddetection and identification approach where the first stage screens forspecific microbe genus/species groups. Then, within the pathogen group,the second stage identifies specific microbe strains/pathotypes.

The initial prescreening stage uses electrochemical measurementsenhanced by nano-scale mechanisms (e.g., increased surface area,nanoconfinement) to recognize hybridization events between the RNAtargets and complementary thiolated DNA, PNA (peptide nucleic acid) orLNA (locked nucleic acid) probes bound to a nanostructured electrodesurface. An active redox probe pair will then be used toelectrochemically detect the hybridization between the target and probeserving to identify specific microbes/pathogens. The redox-basedelectrochemical detection of hybridization events that are detected bydensely-packed probe molecules on nanoporous gold surfaces determinesthe microbe group. The nanoporous surface also acts as a nano-scaleconcentrator via charge-size specific confinement of target molecules ofinterest.

The samples are then analyzed by a final stage designed to providehighly selective identification of a specific strain within a microbe orpathogen group. In an alternative embodiment, this approach may bescaled into a large multiplexed array for high-throughput and rapidscreening. This second stage involves using a nano-scale moveableelectrode to make contact with the target molecules to obtainconductance information. The single nucleic-acid conductancemeasurements reveal target-probe strand match.

It will be appreciated that the subject invention does not requireenzymatic amplification (e.g. PCR). Instead, it relies on RNA, includingbut not limited to mRNA and non-coding RNA (tRNA, rRNA, small RNAs),which microbes naturally amplify via transcription as they express theirgenetic information. Additionally, unique nano-scale enabled phenomenais utilized, including augmented effective surface area for high probedensity and nano-cavity enabled pre-concentration of target species foradditional sensitivity enhancement.

Unlike current optical detection methods, which do not directly detectgenetic information (i.e. require the use of fluorescent markers), thepresent invention directly detects genetic information at the molecularlevel through electrical means.

Aspects of the present invention are founded on recent advances innanotechnology and embodiments of the present invention are ultimatelyintended for hand-held devices which are field-ready for agriculture,food industries and healthcare.

Rapid, efficient, and low cost detection and identification of specificmicrobial pathogens including bacteria, viruses, and fungi is achallenge facing plant and animal (including human) health. The presentinvention addresses this challenge by providing a new technology todetect specific microorganisms within complex environmental media.

In one embodiment, the rapidly increasing number of genome sequencesthat are available for a wide range of microbes are used, includingthose which are pathogens of plants and/or animals. Even closely relatedvariants of a given pathogen species, which may have importantdifferences in host range and/or pathogenicity, differ in their genomesequences. In the subject invention, this information is exploited todetect specific pathogens.

While the majority of current nucleic acid detection approaches targetDNA, the present invention uses “short” and “long” DNA oligomers totarget RNA, as RNA offers many unexploited advantages over DNA.Transcription represents a natural amplification of RNA compared to itsDNA template, thus more RNA is available. Second, controlled degradationof RNA into pieces small enough (even 15 to 20 nucleotides) to use withthe two detection systems proposed here is more facile than it would befor DNA. Lastly, many plant and animal viruses have RNA genomes, and bytargeting RNA, the need to convert RNA sequences into cDNA is obviated.

It is envisioned that the present unified multidisciplinary theme willmake fundamental new contributions to the biological systems studiedhere, and to nano-molecular electronic engineering, and together lead tonew technology for high-throughput, on-site, specific identification ofplant-associated microbes causing plant and human diseases.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe present invention as defined in the claims appended hereto.

Example 1

FIG. 1 shows a schematic diagram of an electrical sensor platform 10 todetect bacteria 12 (i.e. food-borne bacteria) and viruses 14 (i.e. plantviruses) according to an embodiment of the subject invention. Afterfragmentation 16 of the RNA into target 18 and non-target 20 nucleicacid fragments, the fragments are transported to a stage 22 that rapidlyscreens for organism/microbe/pathogen groups using redox-basedelectrochemical detection of hybridization events 26. Thesehybridization events 26 are detected using densely-packed probemolecules (sequences) 28 bound to nanoporous gold electrode surfaces 30and a device for measuring electrical current 24, such as apotentiostat. A magnified image of the stage 22 surface with the goldelectrode surfaces 30 is shown.

This electrochemical approach can be scaled into large multiplexedarrays 32 for high-throughput and rapid screening. Thiolated “probe”sequences 28 that are complementary to the RNA target 18 sequences arebound to a nanoporous electrode surface 30. Hybridization events 26between the target 18 and probe sequences 28 are detected using anelectrochemically active redox probe pairs, such as Ru(NH₃)₆ ³⁺/Fe(CN)₆³⁻. The non-target nucleic acid fragments 20 that do not bind to theprobe sequences 28 settle out.

The samples are next analyzed by a final stage designed to provide, forexample, highly-selective identification of specific strains within amicrobe or pathogen group. It is based on single nucleic-acidconductance measurements that reveal target-probe strand match. Theelectrical read-out capability of such technology enables facileintegration with sensor electronics, and is thus amenable to facileintegration with instrumentation electronics and field-deployabledevices, responding to applications where “sample-to-answer” time iscritical.

The final stage is based on a molecular conductance detection to providegreater specificity. Here, a nanoscale moveable electrode 34 is used tomake repeated contact with a hybridized target-probe pair 36 bound to asecond electrode 38 to obtain conductance information 40. The electricalresistance of short DNA duplexes is extremely sensitive to sequence,length, and even single nucleotide polymorphisms. In this embodiment,the focus is on detecting microbial RNA and identifying specificmicrobial strains. However, this method can be used to detect mutationsin genes that result in specific characteristics or diseases.

This final stage will only need to be implemented in “small” multiplexedarrays 42. Furthermore, in cases where microbe densities areparticularly low (<10 cfu/plant), the single-molecule conductanceapproach will provide additional sensitivity since extremely smallquantities of target molecules 18 will still provide a useable signal.

The prescreening stage is focused on speed and efficiency using a largemultiplexed array format to select for specific organisms based on theirgenomic information. It serves to identify specific organisms, but doesnot resolve small mismatches between target and probe nucleic acidsequences. The final detection stage is optimized for selectivity andsensitivity to uniquely identify specific genetic markers characteristicof individual target organisms.

This effort provides an exciting new paradigm for detecting specific RNAtargets from an extremely small number of sample molecules and has wideranging potential applications.

Example 2

In order to determine whether unique genetic sequences could be detectedwith the approach described, an in silico preliminary study wasundertaken using the nucleic acid sequences of strains 4, 5, and 6 ofgrapevine leafroll-associated viruses, which are 13830, 13384 and 13807nucleotides in length respectively. In silico ribonuclease A-digestionof these sequences, followed by sorting of the residual sequences bylength and comparison between strains was carried out. This analysisidentified 3, 4 and 9 unique residual fragments of lengths ranging from15 to 20 nucleotides from strains 4, 5 and 6 respectively. These 16sequences had an average GC content of 48.5 percent, which shows thathybridization is reasonable.

Example 3

Experimental evidence has been obtained to demonstrate the feasibilityof using single-molecule conductance measurements to detect specific RNAsequences and identify a specific strain of microorganism.

Direct measurement of the electrical conductance of hybrid DNA:RNAmolecules was demonstrated in a liquid environment at thesingle-molecule level by using nanoscale electrodes to directlyinterrogate the electrical properties of the hybrid molecular junction.Notable differences in the conductance properties of DNA:RNA hybridsequences were observed, and it has been experimentally shown that theconductance of such hybrid systems is sensitive to the number of basepairs in the stack, which offers the required specificity to the sensor.

Three DNA probe sequences with lengths of 9, 11, and 13 base pairs weresynthesized with amine terminal groups on both the 5′ and 3′ ends. Theseterminal groups allow direct linkage to atomically sharp goldelectrodes. In an aqueous environment consisting of 100 mM phosphatebuffer solution at pH 7.4, the DNA probe sequence and the complementaryRNA target sequence were hybridized by heating the solution to 80° C.and cooling to room temperature over the course of three hours.

Once the DNA and RNA sequences are hybridized, the conductancemeasurements were performed on three GC-only sequences with lengths of9, 11, and 13 base pairs, as shown in FIG. 2 through FIG. 7. FIG. 2through FIG. 7 show the different aspects of conductance measurements ofDNA:RNA hybrid molecules. FIG. 2 is a schematic illustration 50 of asingle DNA:RNA hybrid 52 bound between two electrodes 54, 56. Theconductance of each hybrid duplex 52 is measured by repeatedly bringingthe nanoscale gold electrodes 54, 56 into contact and withdrawing themin the presence of the buffer solution containing the DNA:RNA hybrids52. Conductance was measured using a transimpedance amplifier.

FIG. 3 shows an example of the N=13 sequence, SEQ. ID. NO. 1, that wasused in the experiments. The DNA probe is terminated with amines so thatit will bind to the gold electrodes. The target RNA sequence wascomplementary to the probe SEQ. ID. NO. 1 sequence of FIG. 3.

If molecules bind between the electrodes 54, 56 during the withdrawalprocess, steps appear in the current vs. displacement trace as shown inFIG. 4. FIG. 4 shows a graph of the current versus displacementresulting from electrode withdrawal. Steps indicate the binding ofmolecules between the two electrodes. By repeating these measurementsthousands of times for each sequence, a statistically significant amountof information is gathered, and a statistical analysis yields the mostlikely conductance of a single molecule. A histogram is constructed fromthousands of current vs. time (or electrode separation) traces forstatistical analysis and yields the most likely conductance of a singleoligonucleotide duplex.

The results for two DNA:RNA hybrids are shown in FIG. 5 and FIG. 6. FIG.5 and FIG. 6 show histograms resulting from a statistical analysis ofcurves similar to that shown in FIG. 4 for the N=9 and N=13 lengthhybrid systems, respectively. There are distinct differences between thestatistical distribution of the conductance associated with each duplex,which demonstrates that break-junction conductance measurements have thecapability of detecting specific RNA sequences.

FIG. 7 shows a graph illustrating conductance vs. length for thesequences that have been studied thus far, demonstrating that theconductance of DNA:RNA hybrids can be measured, and that the conductanceis sensitive to molecular length.

Example 4

The following results demonstrate that the nano-scale features ofnanoporous (np) gold produces a higher electrochemically active areacompared to a planar gold electrode. This enhances the electricalcurrent that is generated in the presence of a nucleic acid, which inturn augments detection sensitivity.

Nanoporous gold (np-Au) samples were synthesized by depositing a thinfilm alloy (˜70% gold and 30% silver by atomic weight) and subsequentlyetching the alloy with nitric acid. This process removed silver andproduces nanoporosity through the re-organization of gold atoms intoligaments. Thermal treatment of np-Au produces various morphologies, asshown in the SEM images of FIG. 8A through FIG. 8D, via ligamentcoarsening. FIG. 8A shows the np-Au with no annealing at roomtemperature. FIG. 8B, FIG. 8C and FIG. 8D show the annealingmorphologies at 200° C., 300° C. and 400° C., respectfully.

The surface area available for reaction is estimated electrochemicallyusing dilute sulfuric acid as the electrolyte. Np—Au films that are notthermally treated yield seven times higher surface area compared to thatof a planar gold surface as shown in FIG. 9.

In order to investigate the effect of DNA probe adsorption onto thesurface, thiolated ss-DNA probes were immobilized onto the np-Au workingelectrode. Ruthenium hexamine chloride and potassium ferrocyanide wereused as the redox markers. In this detection scheme, ruthenium cationswere attracted to the negatively-charged DNA backbone and underwentreduction. The amount of DNA immobilized can be estimated using thisredox marker as shown in FIG. 10. Ferrocyanide regenerates the reducedruthenium cations.

All cited references are incorporated herein by reference in theirentirety. In addition to any other claims, the applicant(s)/inventor(s)claim each and every embodiment of the invention described herein, aswell as any aspect, component, or element of any embodiment describedherein, and any combination of aspects, components or elements of anyembodiment described herein.

From the discussion above it will be appreciated that the invention canbe embodied in various ways, including the following:

1. A method for identifying organisms, the method comprising: providinga target RNA molecule from an organism; contacting, under hybridizingconditions, the target RNA molecule with a complementary nucleic acidprobe, said probe attached to a nanostructured electrode, whereinhybridization of the target RNA molecule and complementary nucleic acidprobe provides an electrochemical current; measuring saidelectrochemical current; and identifying the organism based on saidelectrochemical current measurement.

2. The method of any preceding embodiment, further comprising: providinga hybridized target RNA molecule and complementary nucleic acid probepair; binding the hybridized target RNA molecule and complementarynucleic acid probe pair to a first nanoscale electrode; repeatedlycontacting the hybridized target RNA molecule and complementary nucleicacid probe pair with a second nanoscale electrode; measuringconductance, wherein nucleic acid matches and mismatches between thetarget RNA molecule and complementary nucleic acid probe are detectedbased on said conductance; and further characterizing the organism basedon said nucleic acid matches and mismatches.

3. The method of any preceding embodiment, wherein the organism is amicrobe.

4. The method of any preceding embodiment, wherein the complementarynucleic acid probe is a molecule selected from the group consisting of adeoxyribonucleic acid (DNA), a peptide nucleic acid (PNA) and a lockednucleic acid (LNA).

5. The method of any preceding embodiment, wherein the method isperformed on a large scale multiplex array to allow for a rapiddetection of a plurality of organisms.

6. The method of any preceding embodiment, wherein hybridization betweenthe target RNA molecule and complementary nucleic acid probe is detectedusing an electrochemically active redox probe pair.

7. The method of any preceding embodiment, wherein the electrochemicallyactive redox probe pair comprises Ru(NH₃)₆ ³⁺/Fe(CN)₆ ³⁻.

8. The method of any preceding embodiment: wherein the nanoscaleelectrodes comprise a noble metal; and wherein the complementary nucleicacid probe contains a chemical linker terminal group on both 5′ and 3′ends that attaches to the nanoscale electrodes.

9. The method of any preceding embodiment, wherein the nanostructuredelectrode surface is nanoporous and has a large surface area allowingfor a higher complementary nucleic acid probe density than a planarsurface and a higher electrochemically active platform for higher targetRNA molecule detection sensitivity.

10. The method of any preceding embodiment, wherein naturalamplification of the target RNA molecule is used.

11. The method of any preceding embodiment, wherein the conductance isused to determine a mutation in a gene, said mutation resulting in adisease.

12. A method for identifying the strain of an organism, the methodcomprising: providing a target RNA molecule from an organism;contacting, under hybridizing conditions, the target RNA molecule with acomplementary nucleic acid probe; binding the hybridized target RNAmolecule and complementary nucleic acid probe to a first nanoscaleelectrode; repeatedly contacting the hybridized target RNA fragment andcomplementary nucleic acid probe with a second nanoscale electrode;measuring conductance, wherein nucleic acid matches and mismatchesbetween the target RNA molecule and complementary nucleic acid probe aredetected based said conductance; and further characterizing the organismbased on said nucleic acid matches and mismatches.

13. The method of any preceding embodiment, wherein the organism is amicrobe.

14. The method of any preceding embodiment, wherein the complementarynucleic acid probe is a molecule selected from the group consisting of adeoxyribonucleic acid (DNA), a peptide nucleic acid (PNA) and a lockednucleic acid (LNA).

15. The method of any preceding embodiment: wherein the nanoscaleelectrodes comprise a noble metal; and wherein the complementary nucleicacid probe contains a chemical linker terminal group on both 5′ and 3′ends that attaches to the nanoscale electrodes.

16. An apparatus for detecting and identifying target RNA sequences, theapparatus comprising: a first stage, wherein said first stage comprisesnanostructured noble metal electrodes; nucleic acid probe molecules,wherein said nucleic acid probe molecules are complementary to a targetRNA molecule and wherein said nucleic acid probe molecules are bound tosaid nanostructured noble metal electrodes; a connector for connectingsaid first stage to a potentiostat, wherein hybridization between thetarget RNA molecule and complementary nucleic acid probe is detected bysaid potentiostat using an electrochemically active redox probe pair.

17. The apparatus of any preceding embodiment, further comprising: asecond stage comprising nanoscale electrodes, wherein a hybridizedtarget RNA molecule and complementary nucleic acid probe pair is boundto a first nanoscale electrode and then repeatedly contacted with asecond nanoscale electrode; and a connector for connecting said secondstage to a transimpedance amplifier, said transimpedance amplifierconfigured for receiving a conductance of the target RNA molecule andcomplementary nucleic acid probe.

18. The apparatus of any preceding embodiment, wherein naturalamplification of the target RNA molecule is used.

19. The apparatus of any preceding embodiment, wherein the conductanceis used to determine a mutation in a gene, said mutation resulting in adisease.

20. The apparatus of any preceding embodiment: wherein the nanoscaleelectrodes comprise a noble metal; and wherein the complementary nucleicacid probe contains a chemical linker terminal group on both 5′ and 3′ends that attaches to the nanoscale electrodes.

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

What is claimed is:
 1. An apparatus for detecting and identifying targetRNA sequences, the apparatus comprising: a first stage, wherein saidfirst stage comprises one or more nanostructured noble metal electrodes;nucleic acid probe molecules, wherein said nucleic acid probe moleculesare complementary to a target RNA molecule and wherein said nucleic acidprobe molecules are bound to said nanostructured noble metal electrodesof the first stage; a first connector for connecting said first stage toa potentiostat, wherein hybridization between the target RNA moleculeand complementary nucleic acid probe is detected by said potentiostatusing an electrochemically active redox probe pair; a second stagecomprising one or more nanoscale electrodes; and a second connector forconnecting said second stage to a transimpedance amplifier, saidtransimpedance amplifier configured for receiving a conductance of thetarget RNA molecule and complementary nucleic acid probe; wherein ahybridized target RNA molecule and complementary nucleic acid probe pairbound to said first nanoscale electrode are repeatedly contacted with atleast one second stage nanoscale electrode.
 2. The apparatus of claim 1,wherein natural amplification of the target RNA molecule is used.
 3. Theapparatus of claim 1, wherein the conductance is used to determine amutation in a gene, said mutation resulting in a disease.
 4. Theapparatus of claim 1: wherein the nanoscale electrodes comprise a noblemetal; and wherein the complementary nucleic acid probe contains achemical linker terminal group on both 5′ and 3′ ends that attaches tothe nanoscale electrodes.